Interaction of phenylisothiocyanate with human erythrocyte band 3 protein. II. Topology of phenylisothiocyanate binding sites and influence of p-sulfophenylisothiocyanate on phenylisothiocyanate modification.
The two structurally related probes, the apolar phenylisothiocyanate and the polar, water-soluble p-sulfophenylisothiocyanate, were analysed for their topological interaction with human erythrocyte band 3 protein. Upon thermolytic and peptic digestion of labeled erythrocyte ghosts, the membrane-integrated segments of band 3 protein, the 17,000 and 10,000 dalton peptides, were isolated. At 2 mM initial label concentration, 90% of the hydrophobic probe phenylisothiocyanate was recovered in the 10,000 dalton peptide, the remaining amount of label being associated with the 17,000 dalton fragment. Pretreatment of the membranes with 5 mM p-sulfophenylisothiocyanate followed by labeling with 2 mM phenylisothiocyanate results in a consistent reduction in binding of phenylisothiocyanate by 1 mol/mol isolated band 3 protein. p-Sulfophenylisothiocyanate reportedly binds to the 17,000 dalton fragment (Drickamer, K. (1977), J. Biol. Chem. 252, 6909-6917). The interaction of the polar probe with the membrane protein affects binding of phenylisothiocyanate to the 10,000 dalton peptide by the equivalent of 1 mol/mol isolated peptide. The topological interrelation of the membrane-integrated segments is concluded.
D001457:Anion Exchange Protein 1, Erythrocyte; D001557:Benzenesulfonates / Q000494:pharmacology*; D001798:Blood Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D017879:Isothiocyanates; D008565:Membrane Proteins / Q000378:metabolism*; D010434:Pepsin A; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D013820:Thermolysin; D013861:Thiocyanates / Q000378:metabolism* / Q000494:pharmacology*
D001457:Anion Exchange Protein 1, Erythrocyte; D001557:Benzenesulfonates; D001798:Blood Proteins; D017879:Isothiocyanates; D008565:Membrane Proteins; D010446:Peptide Fragments; D013861:Thiocyanates; C005441:phenylisothiocyanate; C015308:4-isothiocyanatobenzenesulfonate; D010434:Pepsin A; D013820:Thermolysin